Stem cells represent a unique cell type with potential for disease therapy. However, in order to take advantage of this biological potential, we must understand the genetic pathways controlling specification, maintenance and differentiation of progenitor cells. Our laboratory has identified a transcription factor, Foxd3, expressed in a number of embryonic progenitor lineages and in neural crest cells. Perhaps more importantly, Foxd3 functions (both in vivo and in vitro) to maintain stem/progenitor cells in an undifferentiated state as they continue to proliferate. Neural crest-specific deletion of the transcription factor Foxd3 in mice demonstrates a profound requirement for Foxd3 in early events of neural crest maintenance. Virtually all neural crest lineages are adversely affected by the loss of Foxd3, supporting the hypothesis that Foxd3 function is required for maintenance of multipotent stem cell properties of neural crest. Identification of key regulatory genes such as Foxd3 is an important first step in exploiting the potential for manipulation of multipotency in vitro and eventually in vivo for therapeutic goals. In this proposal we will focus on the function of Foxd3 in the maintenance of multipotency in vitro and in vivo: in Aim 1, we will refine the analysis of neural crest stem cells in vitro and the results of deleting Foxd3 from these progenitors with respect to self-renewal and multipotency, and in Aim 2, we will assess neural and glial differentiation of vagal neural crest populations in vivo to precisely define subpopulations of neural crest in which Foxd3 is functioning. These experiments are all focused on understanding the regulation of differentiation of these neural crest progenitors. The mission of the NINDS is to reduce the burden of neurological disease, and neural stem cell therapies are one possible future approach for these diseases. Understanding the molecules controlling neural development will advance the field toward making these treatments a reality. Relevance to Public Health: Accomplishment of these aims will help us to understand the molecular mechanisms regulating neural crest stem/progenitor cells, and eventually, similarities and differences in the molecular regulation of subsets of neural crest progenitors. Knowledge gained here can be directly applied to other stem cell progenitors and will lead to a better understanding of how to manipulate stem cell fate to help facilitate stem cell based therapies in the future.